The present invention relates to a new application of lysozyme dimer. In particular, the present invention relates to a method for the prevention or therapeutic treatment of a disease or bodily condition of a mammal, which disease or condition is directly caused or affected by a hormonal or metabolic disorder such as, in particular, cystic ovarian disease.
In the late 1980s, it was discovered that the isolated dimerized forms of certain enzymes, while substantially retaining the beneficial properties of the corresponding monomers, turned to be by far less toxic than the monomers themselves and in some instances did not even display negative side effects at all when used in therapeutic doses. Antiviral and antibacterial compositions comprising as the active ingredient lysozyme dimer or other dimerized enzymes have been described in WO 89/11294, the entire disclosure of which is incorporated herein by reference. It is reported therein that lysozyme dimer is capable of inhibiting proliferation of a number of bacterial strains cultivated on samples taken from patients, when applied in concentrations of 1.25-20 mg/ml of the culture. It is also reported that the dimer is effective in treating canine parvovirus (CPV) infections when administered orally twice a day at a dose of 1-2 mg/kg of body weight. Later on, further attractive features of lysozyme dimers were found and additional therapeutical applications of the drug were developed, especially for the treatment of bacterial and viral infections as disclosed, for instance, in WO 94/01127, the entire disclosure of which is incorporated herein by reference.
In WO 94/01127, a model theory is presented that can help to understand the different effects observed with the lysozyme dimer. Although the entire mode of action of the lysozyme dimer is not yet fully understood, it appears that there is additional curative capability that cannot be explained by the bacteriolytic activity of the corresponding monomer. The inventors observed certain immunostimulative effects of the dimerized lysozyme, particularly concerning the modulation of cytokine levels. Moreover, from their experiments, they concluded that lysozyme dimer seems to prevent the penetration of bacterial cells by viruses, presumably by blocking certain regions of the outer cell surface and probably comprising virus receptor proteins.
The inventor observed certain immunostimulative effects of the dimerized lysozyme, particularly concerning the modulation of cytokine levels, confirmed by in vitro and in vivo experiments. The lysozyme dimer is able, amongst others, to modulate the synthesis of TNF.DELTA., IL-2, IL-6 and INF.alpha., and to activate phagocytosis and the immunological mechanisms connected therewith. The lysozyme dimer is in particular useful for the treatment and prophylaxis of diseases associated with excessively high levels of TNF.alpha..
The prior art discloses further results obtained in vitro with lysozyme dimer. Particularly, Bartholeyns and Zenebergh (Europ. J. Cancer, Vol. 15, 1979, 85-91) tested dimerized lysozyme for cytostatic activities against liver cancer cells (HCT) in vitro. They observed a 73% .+-.15% inhibition of cancer cell multiplication in the cell culture (ibid., p.89, Table 2).
Surprisingly, except for WO 94/01127, no in vivo experiments with lysozyme dimer are reported so far. It is very strange and astonishing, and up to now waits for explanation, why neither Bartholeyns and Zenebergh nor any other researcher resumed this subject to promote further development of a promising discovery to combat cancer. A comparative showing (FIG. 3) of the purity of lysozyme dimer produced according to the method of Sorrentino et al., Eur. J. Biochem. 124, 183-189 (1982) and of the lysozyme dimer preferably used in the present invention revealed at least one possible reason: high concentrations of by-products such as lysozyme monomer, trimer and tetramer are found in the preparation produced according to Sorrentino et al., whereas the product preferably used in the present invention is highly purified, i.e., contains the desired lysozyme dimer in amounts of up to 90% or more by weight of the total lysozyme fraction of the preparation. A process for the manufacture of such highly purified lysozyme dimer has been described in WO 91/10731, the entire disclosure of which is incorporated herein by reference. This strongly supports the assumption that the purity of the prior art lysozyme dimer was simply not good enough for in vivo experiments and applications because it was known in the art already over 15 years ago that the monomeric form of lysozyme, despite its beneficial antibacterial activity, is rather toxic and can cause inflammations and severe allergies and even toxic shock symptoms.
In light of such circumstances it appears more understandable why no competent researcher including Bartholeyns and Zenebergh--although recommending lysozyme dimer as a promising candidate for further investigations--has carried out further experiments during the past ten to fifteen years to develop lysozyme dimer applications in vivo.
In spite of such lack of research activities of the scientific world possibly due to a prejudice of the art against the use of lysozyme dimer in vivo, the present inventors carried out further research and developmental work to improve the method of production and purification of the dimerized lysozyme and to find in vivo human and animal applications for the product, which led, for instance, to the antiviral and antibacterial and TNF level modulating applications disclosed in WO 94/01127, and the entire disclosure of which is incorporated herein by reference.
It could also be successfully demonstrated that lysozyme dimer compositions display remarkable potency in the inhibition or even total prevention of leukemic cell proliferation in vivo, particularly in the case of virus induced lymphatic leukemia. Further investigations led to the manufacture of pharmaceutical compositions comprising lysozyme dimer as the active component applicable in cases of hair growth disorders, particularly hair growth disorders based on immunological malfunctions or dysfunctions, or in preventing or treating diseases connected with a suppressed immune system. Such compositions, and their applications, are described in U.S. Patent applications Ser. Nos. 08/815,009 and 08/351,375 and International application Ser. No. PCT/EP96/00135, the entire disclosures of which are incorporated herein by reference.
Use of immunostimulants, adjuvants and vaccines offers a wide range of attractive methods for inducing and building up protection against diseases. In this respect, "immunostimulants" refer to compounds that only stimulate non-specific defense mechanisms and protect against diseases. "Immunomodulators" refer to compounds that regulate (or modulate) the defense mechanisms after suppression (or decrease of immunity) of those mechanisms. Such suppression can arise from or be induced by many sources including pollutants, chemotherapeutics, stress, food, temperature changes, and the like. Immunomodulators stabilize the defense mechanisms after the influence of pathogens, and increase cellular and humoral immunity. Some immunomodulators are also able to depress and/or normalize hyperactive defense mechanisms including modulation of cytokine levels. Many immunostimulants are also classified as immunomodulators.
Moreover, based on their knowledge of low toxicity of dimerized lysozyme compared to the monomer. and on the availability of a new, highly purified lysozyme dimer preparation, the present inventors attempted and started anti-cancer trials with lysozyme dimer preparations in vivo, although the prior art did not suggest its use to treat diseases other than bacterial or viral infections.
Further investigative work by the present inventors has also revealed additional advantageous uses of the lysozyme dimer. Recent studies on immunomodulating properties of the lysozyme dimers have shown that it potentiates humoral response in mammals. In particular, the lysozyme dimer has been found to have the unexpected advantage of being capable of positively affecting the primary humoral response of mammals immunized with an antigen after immunosuppression. Such immunomodulating properties of the lysozyme dimer were not taught or suggested by the prior art.